Discovery of dually acting small-molecule inhibitors of cancer-resistance relevant receptor tyrosine kinases EGFR and IGF-1R

Article abstract of DOI:10.1039/c6md00329j

Novel benzo-anellated furo- and pyrrolo[2,3-b]pyridines with a 4-benzylamine substitution have been evaluated as inhibitors of the epidermal growth factor receptor (EGFR). Substituent effects on the determined protein kinase affinity have been discussed based on varied benzylamine residues at the differently substituted molecular scaffolds. Docking studies were carried out in order to explore the potential binding modes of the novel inhibitors. The observed activity data encouraged the measurement of the inhibition of the insulin-like growth factor receptor (IGF-1R), which is known to play an important role in the cancer-resistance development against EGFR inhibitors via receptor heterodimerizations with IGF-1R. We identified novel dual inhibitors of both kinases and report their first cancer cell growth inhibition data.

Full text of DOI:10.1039/c6md00329j

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RESEARCH ARTICLE
Discovery of dually acting small-molecule
inhibitors of cancer-resistance relevant receptor
Cite this: DOI: 10.1039/c6md00329j
tyrosine kinases EGFR and IGF-1R†‡
Cornelius Hempel,a Abdulkarim Najjar,a Frank Totzke,b Christoph Schächtele,b
a
c
a
*
Wolfgang Sippl, Christoph Ritter and Andreas Hilgeroth
Novel benzo-anellated furo- and pyrroloij2,3-b]pyridines with a 4-benzylamine substitution have been eval-
uated as inhibitors of the epidermal growth factor receptor (EGFR). Substituent effects on the determined
protein kinase affinity have been discussed based on varied benzylamine residues at the differently
substituted molecular scaffolds. Docking studies were carried out in order to explore the potential binding
modes of the novel inhibitors. The observed activity data encouraged the measurement of the inhibition of
the insulin-like growth factor receptor (IGF-1R), which is known to play an important role in the cancer-
resistance development against EGFR inhibitors via receptor heterodimerizations with IGF-1R. We identified
novel dual inhibitors of both kinases and report their first cancer cell growth inhibition data.
Received 14th June 2016,
Accepted 2nd August 2016
DOI: 10.1039/c6md00329j
www.rsc.org/medchemcomm
for the development of inhibitors in anticancer therapy.8
Small molecule inhibitors have been developed which address
Introduction
The understanding of cell regulating processes during the
past decades helped to identify deregulated cellular pathways
in cancer cells which result from an abnormal activity of regu-
lating protein kinases.1,2 Such protein kinases can be found
either overexpressed or overactivated in cancer cells so that
the cells show enhanced proliferation rates, escape from apop-
tosis and finally show an aggressive invasive growth into
neighboured tissues.1,3,4 Growth factors play a central role in
such a tumor progression.4 They can be differentiated in ex-
ogenous and cellular factors which contribute not only to the
tumor growth but also to tumor-induced angiogenesis.4
Among the various receptor tyrosine kinases which are in-
volved in cell signalling processes after an exogenous stimu-
lus, the epidermal growth factor receptor (EGFR) is a promi-
nent kinase which contributes to pathogenesis and
progression of various types of cancer.4,5 Recent studies docu-
ment high expression rates for EGFR and the specific EGFR-
binding ligand TGF-α in breast cancer and non-small lung
cancer.4,6,7 So, EGFR represents an important target structure
the intracellular ATP-binding site of EGFR and also monoclo-
nal antibodies were found which block the extracellular li-
gand binding and receptor activation.9,10 Certain problems
with those early small molecule inhibitors have been resis-
tance development resulting from gene mutations.11,12 While
single mutations which cause amino acid exchanges in the
ATP binding regions have been tolerated, mutations in the
encoding exon 20 led to the inactivity of known
inhibitors.13–15 Anticancer therapies using antibodies are criti-
cal because of immense costs and also in those cases resis-
tance developments occur.16,17 Therefore, there is a strong de-
mand for novel, structurally diverse small molecule inhibitors
which show an alternative inhibitor binding and are more ef-
fective concerning the resulting costs for therapy.16
The quinazoline scaffold is a part of prominent EGFR in-
hibitors like erlotinib with
a
4-amino residue.18
Thienopyrimidines have been described as alternative EGFR
inhibitors with partly disappointing cellular activities as far
as reported.19 In those thienopyrimidines, the phenyl part of
the quinazoline scaffold has been replaced with a thiophene
ring, which was substituted with various phenylic residues at-
tached to the 2-position of the thiophene. We synthesized
novel benzo-anellated furoij2,3-b]pyridines as potential novel
EGFR inhibitors. We evaluated their EGFR inhibiting proper-
ties and further varied the molecular scaffold by replacing
the furan residue with a pyrrole residue in the respective
structures. In addition to the inhibition of EGFR, we evalu-
ated the affinities of all compounds towards the insulin-like
growth factor receptor (IGF-1R), which is known to play a
a Department of Pharmaceutical Chemistry, Institute of Pharmacy, Martin Luther
University, Wolfgang-Langenbeck-Strasse 4, 06120 Halle, Germany.
E-mail: andreas.hilgeroth@pharmazie.uni-halle.de; Tel: +49 345 5525168
b ProQinase GmbH Freiburg, Breisacher Straße 117, 79106 Freiburg, Germany
c Department of Clinical Pharmacy, Institute of Pharmacy, Ernst Moritz Arndt
University of Greifswald, Friedrich-Ludwig-Jahn-Strasse 17, 17489 Greifswald,
Germany
† The authors declare no competing interests.
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6md00329j
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central role in EGFR-mediated cancer-resistances due to the
heterodimerization of EGFR with IGF-1R as will be discussed
below. In this way, we identified the first promising dual
EGFR and IGF-1R inhibitors for which their first cellular anti-
cancer screening data will be reported.
ries 7 under reflux conditions. The given benzylamine com-
pound series was finally treated with tinIJII) chloride in hydro-
chloric acid to yield the 6-amino compound series
8 (Scheme 1).
The benzo-anellated pyrroloij2,3-b]pyridine 10 was formed
from (2-pyridyl) benzotriazole (9) in a polyphosphoric acid
(PPA)-catalyzed reaction. The benzotriazole 9 resulted from
the reaction of 2-bromopyridine and benzotriazole in toluene
under reflux conditions. The 4-chloro function had been in-
troduced in derivative 11 after activation of the nitrogen of
compound 10 using hydrogen peroxide in acetic acid to give
the N-oxide that reacted with phosphorus oxychloride to give
the 4-chloro derivative 11. The 6-nitro function was intro-
duced similarly to compound 7 with fuming nitric acid. The
4-benzylamino compound series 13 resulted from the direct
heating of the 4-chloro-6-nitro compound 12 with the
benzylamines at varying temperatures. Finally, the 6-amino
compound series 14 formed after treatment of the 6-nitro
function of the benzylamine substituted compounds 13 with
tinIJII) chloride under hydrochloric acid conditions
(Scheme 2).
Results and discussion
The benzo-anellated furoij2,3-b]pyridine scaffold 4 was formed
from 3-amino-2-phenoxy-pyridine (3) in a copper-catalyzed re-
action after treatment with sodium nitrite in sulfuric acid.
The precursor of compound 3 with a nitro function at the
3-position of the pyridine had been treated with hydrogen in
a palladium-catalyzed reaction to reduce the nitro function. The
2-phenoxy group of precursor 2 had been introduced by treat-
ment of the commercially available 2-chloro-3-nitropyridine (1)
with phenol using sodium hydride. The 4-chloro substitution
in compound 5 had been introduced using phosphorus
oxychloride after activation of the nitrogen as N-oxide with
meta-chloroperbenzoic acid (mCPBA), so that the chlorine
atom was preferably introduced into the 4-position of the ac-
tivated scaffold. The 6-nitro function had been selectively in-
troduced into compound 5 with fuming nitric acid at low
temperature and then the varying benzylamines reacted with
the given 6-nitro compound 6 to form the target structure se-
The EGFR-wild type (wt) affinities have been determined
in an ATP competition assay as described in the ESI.† In
short, the reduced amount of phosphorylated EGFR substrate
has been measured using a scintillation technique under
Scheme 1 Formation of the benzo-anellated furoij2,3-b]pyridine 4 with reaction conditions and yields: (1) NaH, phenol, toluene, reflux, 89%; (2)
Pd/BaSO4, H2, 98%; (3) H2SO4, NaNO2, 0 °C, Cu, 55 °C, acetone, 22%; (4) mCPBA, CHCl3, reflux, 82%; (5) POCl3, CHCl3, reflux, 69%; (6) HNO3,
0 °C, 97%. Derivatization reaction to target structure series 7 and the final reaction to compound series 8 with reaction conditions and yields: (7)
benzylamine cpd., reflux, 1.4–57%; (8) SnCl2, HCl, reflux, 18–96%.
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Scheme 2 Formation of the benzo-anellated pyrroloij2,3-b]pyridine 10 with reaction conditions and yields: (1) toluene, reflux, 97%; (2) PPA, 170
°C, 36%; (3) H2O2, HAc, reflux, 73%; (4) POCl3, DMF, 0 °C, 58%; (5) HNO3, 0 °C, 75%. Derivatization reaction to target structure series 13 and the fi-
nal reaction to compound series 14 with reaction conditions and yields: (6) benzylamine cpd., 145–160 °C; 43–90%; (7) SnCl2, HCl, reflux; 41–71%.
varied inhibitor concentrations. From the resulting IC50
values, the affinity constants have been calculated. For the
Table 1 Inhibitory activities towards EGFR-wt and IGF-1R determined as
3-methoxybenzylamine derivative 7a, we found a low micro-
molar affinity towards EGFR (Table 1).
Ki values of our benzylamine substituted target compounds 7a–h, 8a–g,
13a–c and 14a–c
When the methoxy function was moved to the 4-position
of the benzylamine, as shown in derivative 7b, the affinity
slightly increased. The 3-chloro benzylamine compound 7c
showed an improved affinity compared to the 3-methoxy
compound. If placed into the 4-position of the benzylamine,
as shown in compound 7d, the affinity was lowered. Next we
investigated the influence of a bromo substituent, as shown
in compounds 7e and 7f. The 3-bromo substitution was com-
parably more favourable than the investigated chloro and
methoxy substitutions leading to a submicromolar affinity in
compound 7e towards EGFR. When moving the bromo sub-
stituent to the 4-position, we found a similar decrease in the
affinity for compound 7f to what has been stated for the
chloro substituent movement. Then we tested derivative 7g,
which has a 3-amino substituent. The compound showed the
poorest affinity among all derivatives so far. If replaced with
Ki valuea
Cpd.
R1
R2
EGFR-wt (μM)
IGF-1R (μM)
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8d
8e
OMe
H
Cl
H
Br
H
OMe
H
Cl
H
Br
H
H
H
OMe
H
Cl
H
2.74 0.07
2.05 0.02
1.36 0.03
2.52 0.12
0.68 0.02
0.81 0.01
3.59 0.06
0.93 0.04
0.17 0.01
n.a.b
4.56 0.04
3.40 0.02
2.85 0.28
3.54 0.08
2.61 0.06
2.87 0.24
6.18 0.11
2.39 0.04
0.54 0.05
n.a.b
H
NH2
NO2
OMe
H
Cl
H
Br
H
NH2
OMe
Cl
0.40 0.04
4.60 0.05
n.a.b
n.a.b
0.23 0.02
n.a.b
n.a.b
8f
8g
Br
H
n.a.b
0.94 0.03
0.33 0.04
0.08 0.02
0.11 0.02
0.32 0.01
0.14 0.01
0.11 0.02
8.93 0.98
0.89 0.02
0.28 0.04
0.32 0.07
3.56 0.01
1.92 0.02
2.10 0.03
13a
13b
13c
14a
14b
14c
—
—
—
—
—
—
a
nitro function, as in derivative 7h, we found a
Br
OMe
Cl
submicromolar affinity towards EGFR. Summarizing the
given results we can state that a 3-substitution in the
benzylamine residue is more favourable than a 4-substitution.
Best affinities have been found for the 3-bromo and the
Br
a
b
Mean of two determinations. Not active, IC50 value >1000 μM.
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3-nitro substitution reactions, whereas the 3-amino substi-
tution is unfavourable.
So we identified most favourable 3-benzylamine
substituted derivatives with highest affinities towards EGFR
in the 6-amino furopyridines series on one hand and in the
6-nitro pyrrolopyridine series on the other hand reaching
nanomolar ranges.
Then we investigated our 6-amino substituted series 8.
The 3-methoxy substitution in derivative 8a led to a more
than fifteenfold higher affinity towards EGFR than the same
substitution in compound 7a. However, if moved to the 4-po-
sition, the resulting compound 8b was found inactive as an
EGFR inhibitor. A similar strong difference in activity was
found in the case of the chloro-substituted compounds 8c
Resistance developments of the established EGFR inhibi-
tors have recently been described to result from the hetero-
dimerization of one EGFR receptor half with other recep-
tors.20,21 After binding of the extracellular ligand, one EGFR
receptor half normally undergoes homodimerization to give a
functional receptor dimer that is autophosphorylated at the
cytoplasmatic tyrosine residues.22,23 In the following cell sig-
nalling proteins dock to the phosphorylated receptor site and
are activated by phosphorylation to proceed in the cell signal-
ling process.21,24 Alternatively, to the primary homo-
dimerization one EGFR receptor half may dimerize with an-
other receptor.20,21 In that case an inhibiting compound may
not prevent the EGFR activity and resistance results. That
kind of anticancer drug resistance mechanism has been de-
scribed for erlotinib as a consequence of heterodimerization
with IGF-1R in an aggressive type of non-small lung cancer.20
So there has been strong interest to develop dually acting
inhibitors of both EGFR and IGF-1R. Such novel inhibitors
ideally have a novel molecular scaffold different from the
established EGFR inhibitors so that potential resistance de-
velopment as result of EGFR mutations is lowered. Our novel
furo- and pyrrolopyridines own such a novel molecular scaf-
fold and so we investigated them to act not only as EGFR in-
hibitors as demonstrated, but also as IGF-1R inhibitors to
compete with the second EGFR resistance mechanism of re-
ceptor heterodimerization.
First we investigated the furopyridines 7 and 8 as IGF-1R
inhibitors in the ATP competition assay as described for the
inhibition of EGFR. Compound 7a showed a lower micromo-
lar affinity towards IGF-1R, which slightly improved in com-
pound 7b with the 4-methoxy substitution. In the case of the
varying chloro substitutions in derivatives 7c and 7d, we
found a slightly better affinity for the 3-chloro derivative 7c
whereas the 4-chloro compound 7d was as active as the
4-methoxy compound 7b. The compounds with bromo sub-
stituents showed best results both in the 3- and the 4-posi-
tions if compared to the varied methoxy and chloro positions.
The 3-amino function in compound 7g showed decreased af-
finities compared to the corresponding bromo function. The
3-nitro derivative 7h was the best IGF-1R inhibitor of this
class of 6-nitro furopyridines. Favourably, that compound
was also the best EGFR inhibitor within this group of 6-nitro
substituted compounds, so that we identified a first dual
inhibitor.
and 8d. While the 3-chloro substitution resulted in
a
favourable submicromolar affinity, the 4-chloro substitution
caused a loss of affinity. So we found the same tendency that
a 3-substitution with either methoxy or chloro was favourable
reaching low submicromolar ranges, whereas the
4-substitution led to inactive compounds. The tendency was
also confirmed for the bromo-substituted compounds. The
3-bromo substitution in compound 8e led to a lower micro-
molar affinity whereas the 4-bromo substituted compound 8f
was not active. Finally, the 3-amino substitution in derivative
8g was much more favourable than that of the 6-nitro com-
pound 7g.
The given results prove that the 3-benzylamine substitu-
tions are more favourable in the 6-amino compound series
8 than in the 6-nitro series 7, whereas benzylamine substitu-
tions in the 4-position are not tolerated in the 6-amino com-
pound series 8. However, also in the 6-nitro series 7, de-
creased affinities have been found for the 4-substituted
benzylamino compounds when compared to those
3-substituted benzylamine derivatives.
Next we investigated the varied benzylamine substitutions
in our pyrrolopyridine series 13. The 3-methoxy substitution
of derivative 13a was more favourable than that of the
furopyridine compound 7a. The 3-chloro function in com-
pound 13b led to a more than fifteenfold higher affinity to-
wards EGFR than that in the corresponding furopyridine de-
rivative 7c. This compound showed a nanomolar affinity with
a Ki value of 82 nM. Also the 3-bromo compound 13c showed
a higher affinity towards EGFR than the respective compound
7e. So we found the investigated 3-benzylamine substituted
pyrrolopyridines with the 6-nitro functions to be more active
as EGFR inhibitors than the furopyridines reaching nano-
molar affinity ranges.
With the 6-amino function, we found almost the same af-
finities for the 3-methoxy benzylamino substitution in com-
pound 14a as in derivative 13a. However, the 3-chloro substi-
tution in the 6-amino compound 14b was less favourable
than that in the 6-nitro derivative 13b. The 3-bromo substitu-
tion in compound 14c with the 6-amino function was as
favourable as in the 6-nitro compound 13c. If we compare
the pyrrolopyridines with the 6-amino function with the cor-
responding furopyridines, we found the 3-chloro and
3-bromo benzylamine residues to be more favourable in the
pyrrolopyridines. However, some higher affinity towards
EGFR resulted for the 3-methoxy benzylamine substitution in
the furopyridine 8a with the 6-amino function if compared to
the pyrrolopyridine 14a.
Then we investigated the next group of furopyridines with
the 6-amino function as IGF-1R inhibitors. The 3-methoxy
function in derivative 8a was very favourable reaching
submicromolar affinity ranges. When the methoxy function
was moved to the 4-position, the activity of the resulting com-
pound 8b was lost. A similar result was found for the varying
chloro substitutions in compounds 8c and 8d. While the
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3-chloro derivative 8c showed a micromolar activity, the
4-chloro compound 8d was found inactive. However, the sub-
stitution of the bigger bromine atom led to inactive com-
pounds both in the 3- and the 4-positions of the benzylamine
residues in compounds 8e and 8f. For the 3-amino substitu-
tion in derivative 8g, we found a lowered IGF-1R affinity com-
pared to the 3-methoxy and 3-chloro substituted compounds
8a and 8c. In our second group of furopyridines, we found a
tendency similar to our first group with the 6-nitro function
concerning the IGF-1R affinity. Obviously, the 4-substituted
benzylamino function is less or unfavourable for substituents
as demonstrated. With the 3-methoxy benzylamine com-
pound 8a, we identified our second dual inhibitor of both
EGFR and IGF-1R with mainly improved affinities if com-
pared to the best dual inhibitor of the 6-nitro furopyridine se-
ries 7h.
Finally, we determined the IGF-1R inhibitory activities of
our pyrrolopyridines starting with the 6-nitro substituted
compounds 13a–c. All compounds showed submicromolar
affinities with the 3-chloro substitution of derivative 13b be-
ing the best one for the inhibitory activities. The 3-methoxy
substitution of compound 13a was less favourable and the
3-bromo substitution in derivative 13c was just slightly
poorer than the 3-chloro function substitution. With a
substituent ranking from chloro to bromo and, finally,
methoxy of decreased affinities towards IGF-1R, we found a
similar tendency in activities for the furo- and pyrrolopyridine
series of the 6-nitro substitution. The pyrrolopyridines,
however, showed an up to tenfold higher affinity towards
IGF-1R than the furopyridines. In the compound series with
the 6-amino pyrrolopyridines 14a–c, the ranking of the
3-benzylamino acids was the same as that of the 6-nitro series
with the 3-chloro substituent of compound 14b being the
best one, followed by the bromo substituent in derivative
14c and, finally, the methoxy residue in compound 14a. How-
ever the affinities of compounds 14a–c were about sevenfold
lower than those of the 6-nitro series 13a–c. All 6-nitro deriva-
tives 13a–c were dual inhibitors of EGFR and IGF-1R with
compound 13b being the best one with a nanomolar EGFR
inhibitory activity and a lower submicromolar affinity to-
wards IGF-1R.
In order to provide a potential rationalisation for the
detected in vitro activity, docking studies were done for the
two kinases EGFR and IGF-1R in their active form. The inhib-
itors were docked into the ATP binding pocket using the
GOLD docking program (see the ESI†) and default docking
settings. The benzo-anellated furoij2,3-b]pyridine and benzo-
anellated pyrroloij2,3-b]pyridine scaffolds are mimicking the
adenine ring of ATP and represent hinge-binding motifs. The
docking results show that the active inhibitors are able to
form a hydrogen bond between the pyridine nitrogen and the
backbone NH of the hinge residue Met793 (EGFR), and
Met1082 (IGF-1R), respectively (Fig. 1a–d). The different ori-
entation results from the varying size of the gatekeeper resi-
due (Thr790 in EGFR and Met1079 in IGF-1R). The different
gatekeeper residues and the size of the hydrophobic back
Fig. 1 Docking results for EGFR. a) The pyridine ring is accepting a
hydrogen bond from the hinge backbone (NH of Met793). The 3-meta-
bromobenzyl ring of 8e (green) is placed in the hydrophobic pocket
nearby the gatekeeper (formed by Met766, Leu788 and Thr790),
whereas the 6-amino group is donating a hydrogen bond to the back-
bone of the P-loop residue Leu718. b) A similar binding mode is ob-
served for the pyrrolopyridine derivatives (e.g. the one shown in or-
ange is 14c).
A second hydrogen bond is observed between the
pyrrolo NH and the carbonyl group of Met793. Hydrogen bond dis-
tances are given in Å. c) In the case of the 6-nitro derivatives (e.g. the
one shown in magenta is 7e and in pink is 7f), both 3-meta and
4-para-substituted benzyl groups can be accommodated in the hydro-
phobic pocket nearby the gatekeeper. d) Molecular surface of the
EGFR binding pocket – hydrophobic parts are colored green while hy-
drophilic regions are colored magenta. The docked inhibitors 7e and 7f
are shown.
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pocket are mainly responsible for the observed selectivity of
some of the inhibitors.
The docking results for EGFR show that the pyridine ring
of the inhibitors from the four series is accepting a hydrogen
bond from the hinge backbone (NH of Met793). In the case
of the 6-amino substituted derivatives (series 8 and 14), a hy-
drogen bond to the backbone of the P-loop residue Leu718 is
further fixing the position of the inhibitors. In this position,
a substitution at the benzyl ring is only sterically possible in
the 3-meta position resulting in favourable van der Waals in-
teractions with Met766, Leu788 and Thr790 (Fig. 1a and b).
In the case of the 4-para substitution (i.e. 8b, 8d, 8f), a steric
clash with Met766 and Leu788 would occur. In the case of
the 6-nitro derivatives, this hydrogen bond is not possible
resulting in a position that is slightly moved out of the
pocket (Fig. 1c and d). As a consequence, both 3-meta- and
4-para-substituted benzyl groups can be accommodated in
the hydrophobic pocket nearby the gatekeeper.
The docking results for IGF-1R show that similar hydrogen
bonds exist between the furopyridine and pyrrolopyridine
rings with the hinge region residues. The hydrophobic pocket
nearby the gatekeeper Met1079 shows a different size from
the pocket in EGFR. However, also here the docking of
3-meta-substituted benzyl derivatives gave more favourable re-
sults as shown for compound 13c in Fig. 2a. In the case of
the 4-para-substituted benzyl derivatives, the hydrophobic
ring is adopting a different conformation that is less buried
(Fig. 2b). This might explain the inactivity of some of the
4-para-substituted compounds (i.e. 8b, 8d, 8f).
Next we initially screened the anticancer activity of our
most promising compounds in various cancer cells lines. As
discussed above, EGFR and its exogenous ligands have been
reported to be predominantly overexpressed in breast cancer
and non-small lung cancer. EGFR overexpression has been
reported to occur in 30% of the breast cancer types.25 We se-
lected cell lines BT-549 and HS 578T as two breast cancer cell
lines with proven EGFR-expression rates.25,26 Furthermore,
two non-small lung cancer cell lines have been selected for
our initial screening. For both cell lines, an EGFR-
overexpression has been documented.27 Cellular growth in-
hibitory activity has been determined for one given concen-
tration to estimate the benefit of an effective EGFR inhibition
for our compounds. We selected the dual EGFR/IGF-1R inhib-
itors 8a and 13b and the only EGFR-inhibiting compound 8e.
The results for percent growth inhibition at the given screen-
ing concentration using the sulforhodamine fluorescence as-
say are shown in Table 2.
Fig. 2 Docking results for IGF-1R. a) The pyridine ring is accepting a
hydrogen bond from the hinge backbone (NH of Met1082). A second
hydrogen bond is observed between the pyrrolo NH and the carbonyl
group of Met1082. The 3-meta-bromobenzyl ring of 13c (orange) is
placed nearby the gatekeeper residue. b) Docking results for the
furopyridine derivatives 7e (magenta) and 7f (pink). In the case of the
4-para-substituted benzyl derivative 7f, the hydrophobic benzyl ring is
adopting a different position that is less buried.
in the activity of our submicromolar inhibitors 8a and 8e to
the nanomolar inhibitor 13b. We found a growth inhibition
of 18% and 23% for our non IGF-1R inhibiting EGFR inhibi-
tor 8e. The slightly improved EGFR inhibitor with the
submicromolar IGF-1R affinity of compound 8a resulted in a
Table 2 Tumor cell growth inhibition at a concentration of 10 μM of the
respective inhibitors 8a, 8e and 13b in screened breast cancer and non-
small lung cancer cell lines
In the breast cancer cell line BT-549, we found growth in-
hibition for our compounds ranging from 29% to 43%. In
the other breast cancer cell line HS 578T, we found a differ-
ence in the compound activities. Our submicromolar EGFR
inhibitors 8a and 8e showed a poorer activity with a growth
inhibition of just 16%, whereas our nanomolar EGFR inhibi-
tor 13b with the best IGF-1R inhibiting properties resulted in
a growth inhibition of 82%.28 In the non-small lung cancer
cell lines HOP-62 and NCI-H460, we found similar differences
Compound growth inhibition (%)
Cancer
cell line
8a
8e
13b
Breast cancer
BT-549
HS 578T
43
16
29
16
30
82
Non-small lung cancer
HOP-62
NCI-H460
28
29
18
23
89
73
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growth inhibition of 28% and 29%. Our nanomolar EGFR in-
hibitor 13b with the comparably best IGF-1R affinity caused a
growth inhibition of 89% and 73%.
4 N. Normanno, A. De Luca, C. Bianco, L. Strizzi, M. Mancino,
M. R. Macello, A. Carotenuto, G. De Feo, F. Caponigro and
D. S. Salomon, Gene, 2006, 366, 2.
Our first screening results prove that our inhibitor 13b
with the best dual affinities towards EGFR and IGF-1R may
become a promising drug candidate to combat cell growth in
non-small lung cancer.
5 M. K. Needergard, C. J. Hedegaard and H. S. Poulsen,
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It can be concluded that our novel furo- and pyrrolopyridines
show EGFR and IGF-1R inhibiting activities which depend on
both the benzylamino and the molecular scaffold substitu-
tions. For the newly synthesized compound 15 of the
furopyridine type with a 4-methoxybenzylamine substituent
and without a substituent in the 6-position of the molecular
scaffold,29 we characterized the EGFR and IGF-1R inhibiting
properties, yielding 1.43 μM for EGFR-wt and 1.03 μM for
IGF-1R. That compound could have been screened for a first
selective kinase inhibition in a novel project. The compound
has been investigated to inhibit two EGFR mutants EGFR-
T790M and -L858R with both determined inhibition values
>100 μM, so that the compound was more sensitive to the
wild type EGFR. Further screening efforts included EGFR-
related kinases HER2 and HER4 as well as the related recep-
tor tyrosine kinases JAK2 and 3 and TIE-2 with similar values
>100 μM. If screened against a few other kinases from partly
different kinase families VEGFR2 and 3, PDGFR-β and GSK-
3β, we found no activity for the compound with inhibition
values >100 μM as determined. So we can document the first
selectivity to EGFR-wt and IGF-1R inhibition for this novel
compound class.
In our discussed structure–activity compound studies, we
identified the first dual inhibitors of both kinases with
submicromolar and nanomolar affinities, which showed
growth inhibition in EGFR-related breast and non-small lung
cancer cell lines. With reference to the reported EGFR- and
IGF-1R heterodimerization in such a non-small-lung cancer
tumor, our dual inhibitor 13b characterized to inhibit non-
small lung cancer cell growth may become an attractive com-
pound for further drug development.
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Acknowledgements
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The authors acknowledge the financial support of their work
by the DFG (German Research Foundation) to Cornelius
Hempel and to Andreas Hilgeroth within the project HI 687/
10-1.
26 J. L. Martin, H. C. de Silva, M. Z. Lin, C. D. Scott and R. C.
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28 The increased inhibitory activities of compound 13b may be
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ferred to in the literature.26
A
stimulation of EGFR
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expression had been documented under an insulin-like
MedChemComm
29 N-(4-Methoxybenzyl)benzofuroij2,3-b]pyridine 15. Yield 0.08 g
(61%); beige solid; mp 148–154 °C; 1H NMR (DMSO-d6) δ
3.09 (s, 3H, CH3), 4.56 (d, J = 6.0 Hz, 2H, CH2), 6.49 (d, J =
5.9 Hz, 1H, 3-H), 6.88 (d, J = 8.4 Hz, 2H, 2′-, 6′-H), 7.33 (d, J
= 8.4 Hz, 2H, 3′-, 5′-H), 7.37–7.48 (m, 3H, 6-, 7-, NH), 7.63 (d,
J = 7.9 Hz, 1H, 8-H), 7.91 (d, J = 5.9 Hz, 1H, 2-H), 8.43 (d, J =
7.5 Hz, 1H, 5-H); MS (EI), m/z = 304 [M+]; IR (KBr): 3319,
1604, 1584, 1514, 1467, 746 cm−1. Anal. (C19H16N2O2) Calc. C
75.0, H 5.3, N 9.2; found C 75.4, H 5.4, N 8.8.
growth factor-binding protein which is known to act also
via IGF-1R,26 so that an IGF-1R inhibition may increase
an EGFR-inhibiting effect. The only EGFR-inhibiting com-
pound gefitinib had been reported to result in an only
20% cellular growth inhibition of HS 578T at a concen-
tration of 4 μM.26 That comparison may support the fa-
vour of a dual EGFR/IGF-1R inhibition as shown for our
compound 13b.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2016